Methyltransferase capable of catalytically generating emoditin-3-methyl ether and application of methyltransferase

Abstract

The invention relates to methyltransferase capable of catalyzing methylation of anthraquinone compounds, the amino acid sequence of the methyltransferase is as shown in SEQ ID NO: 2, and the invention also provides application of the methyltransferase in production of methylated anthraquinone compounds such as emoditin-3-methyl ether. According to the present invention, the new methyltransferase is excavated, such that the anthraquinone compound can be methylated, and the emodin can be converted into the emoditin-3-methyl ether; according to the invention, an engineering bacterium capable of producing the emodin-3-methyl ether is constructed by utilizing the coding gene of the emodin-3-methyl ether transferase, a methyltransferase library capable of being used for emodin methylation is widened, and a new way is provided for construction of an emodin-3-methyl ether cell factory.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering, and relates to a methyltransferase capable of catalyzing the methylation of anthraquinone compounds such as rhubarb, a coding gene thereof and an application thereof. Background Art

[0002] The continuous use of chemical pesticides will cause serious environmental pollution. Botanical pesticides are derived from natural plants, have low toxicity to humans and animals, and have good environmental compatibility, and have become a new development hotspot in the global pesticide industry.

[0003] Rhamnosin and its derivative Rhamnosin-3-methyl ether (CAS registration number: 521-61-9) have multiple biological activities such as antibacterial, anti-inflammatory, anti-cancer, antitussive, and free radical scavenging. Medical research shows that Rhamnosin-3-methyl ether has good control effects on powdery mildew, downy mildew, gray mold and anthracnose, and has been developed into a low-toxic and environmentally friendly biological pesticide product for disease control in green agriculture and organic vegetables. However, the traditional production method of plant-derived compounds has harsh growth conditions and long cycles, and the complex components will increase the cost of separation and purification. Rhamnosin-3-methyl ether has a low content in plants, only 0.1%, and the cost of direct separation and extraction is high. At the same time, it is difficult to achieve stable mass production due to the harsh growth conditions, long growth cycle, and environmental factors of plants, which seriously limits the agricultural promotion of Rhamnosin-3-methyl ether fungicide, and the market share has not been high.

[0004] Therefore, reconstructing the microbial cell factory of rhamnosin-3-methyl ether based on the concept of synthetic biology is of great significance for breaking through the shackles of production capacity and quality and expanding market share. At present, there are inventions that use genetic engineering strategies to introduce exogenous rhamnosin 3-OH O-methyltransferase genes to construct a genetically engineered strain that efficiently accumulates rhamnosin-3-methyl ether, and develop a new method for preparing plant-derived biofungicide rhamnosin-3-methyl ether through fermentation synthesis.

[0005] In order to realize the above method, it is necessary to discover new methyltransferases. Summary of the invention

[0006] During the research, we discovered a new methyltransferase that can methylate the hydroxyl group at the C3 position of anthraquinone compounds to produce methoxyl group, and verified its function through in vivo knockout and in vitro enzyme activity, proving that it has methylation function.

[0007] On this basis, the present invention provides a methyltransferase that can catalyze rhamnosin to generate rhamnosin-3-methyl ether, and the amino acid sequence is shown in SEQ ID NO:2.

[0008] The present invention also provides a nucleic acid encoding the above methyltransferase.

[0009] In a specific embodiment, the sequence of the nucleic acid is shown in SEQ ID NO:1.

[0010] The present invention also provides application of the methyltransferase in methylating anthraquinone compounds.

[0011] The present invention also provides a method for preparing methylated anthraquinone compounds, comprising the steps of mixing the anthraquinone compounds to be methylated with the above-mentioned methyltransferase and a methyl donor and carrying out a catalytic reaction.

[0012] In a specific embodiment, the catalytic reaction is performed in vitro.

[0013] In a specific embodiment, the catalytic reaction is performed in vivo by expressing the methyltransferase in cells containing the anthraquinone compound to be methylated.

[0014] In a specific embodiment, the methylated anthraquinone compound is rhein-3-methyl ether, and the cell is a cell capable of accumulating rhein.

[0015] In a specific embodiment, the cell is Aspergillus terreus capable of accumulating rhein.

[0016] In a specific embodiment, the gedA gene is knocked out in the Aspergillus terreus.

[0017] In a specific embodiment, the amino acid sequence encoded by the gedA gene is shown in SEQ ID NO:3.

[0018] The present invention also provides a cell prepared by the above method and capable of producing methylated anthraquinone compounds.

[0019] The present invention has discovered a new methyltransferase that can methylate anthraquinone compounds and convert rhein into rhein-3-methyl ether. The present invention uses the coding gene of the enzyme to construct an engineered bacterium that can produce rhein-3-methyl ether, broadens the methyltransferase library that can be used for rhein methylation, and provides a new way to construct a rhein-3-methyl ether cell factory. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Genomic PCR verification results of transformants obtained by knocking out the ngoD gene.

[0021] Figure 2 Shown are the HPLC analysis results of the mutant strain MKAJ-ΔngoD and the control strain MKAJ.

[0022] Figure 3This is the SDS-PAGE analysis result of protein NgoD.

[0023] Figure 4 The results of in vitro enzyme activity reaction of NgoD catalyzing the methylation of compound 2. Among them, (I) is the standard of compound 1; (II) is the standard of compound 2; (III) is the sample of normal reaction; (IV) is the control group without enzyme; (V) is the control group without donor; (VI) is the control group without substrate.

[0024] Figure 5 The results of in vitro enzyme activity reaction catalyzing the methylation of rhamnosin. Among them, (I) is the rhamnosin standard; (II) is the rhamnosin-3-methyl ether standard; (III) is the sample of normal reaction; (IV) is the control group without enzyme; (V) is the control group without donor; (VI) is the control group without substrate.

[0025] Figure 6 Schematic diagram of the three expression elements for expressing ngoD at the gedA site in Aspergillus terreus and knocking out gedA at the same time.

[0026] Figure 7 The results of PCR verification of genome extraction from Aspergillus terreus transformants. A is the result of PCR verification of genome extraction from transformants obtained by transferring the expression element UgedA-ngoD-Tpgk-ptrA-DgedA into Aspergillus terreus HZ04; B is the result of PCR verification of genome extraction from transformants obtained by transferring the expression element UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedA into Aspergillus terreus HZ04; C is the result of PCR verification of genome extraction from transformants obtained by transferring the expression element UgedA-PgpdAt-ngoD-Tpgk-ptrA-DgedA into Aspergillus terreus HZ04.

[0027] Figure 8 HPLC analysis of fermentation products from transformants derived from three strategies. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0029] 1. Functional verification of the methyltransferase NgoD in the fungus Echinopsium spinulosum MEFC009

[0030] By analyzing the genome sequence and fermentation product analysis of the fungus Coelocystis acanthosporium MEFC009 of the genus Coleoptilospora, it was speculated that the gene ngoD (the nucleic acid sequence is shown in SEQ ID NO: 1, and the encoded protein sequence is shown in SEQ ID NO: 2) may have the function of a methyltransferase. In order to verify its function, the gene was knocked out in the strain MKAJ. The method is as follows:

[0031] The genome of wild-type Coleophoma sp.MEFC009 was used as a template to amplify the upstream sequence U-ngoD of ngoD with a size of about 1201 bp and the downstream sequence D-ngoD of ngoD with a size of 1200 bp. The hygromycin resistance screening fragment hph fragment (size of 2183 bp), the upstream sequence U-ngoD and the downstream sequence D-ngoD were fused by fusion PCR, and then the fusion product was used as a template to amplify the knockout expression element UngoD-hph-DngoD with a size of 4257 bp using nested primers U-ngoD-CS-F / D-ngoD-CS-R.

[0032] Taking MKAJ as the starting strain, first take a small amount of mycelium from the PDA plate, break it with a handheld homogenizer, take 1mL of seed liquid and inoculate it into 50mL of ME seed medium, and culture it in a 250mL Erlenmeyer flask at 220rpm and 25℃. After 2 days, collect the mycelium by centrifugation (5000rpm, 4℃, 5min). The mycelium is broken again with a homogenizer, and 2mL of seed liquid is inoculated into 50mL of ME seed medium. Culture it under the same conditions for 1 day, pour the medium and mycelium into a 50mL sterile centrifuge tube, and collect the mycelium by centrifugation at 5000rpm. Wash the mycelium 5 times with 0.6M MgSO4. Weigh 1g of mycelium, add 10mL of ME enzymatic solution (filtered and sterilized through a 0.22μm sterile filter), and treat it at 30℃ and 130rpm for 4h. Filter the above protoplast reaction solution through a sterile magic filter cloth, and collect the protoplasts by centrifugation at 5000rpm and 4℃. Wash once with pre-cooled STC, resuspend the protoplasts in pre-cooled STC, and adjust the protoplast concentration to 10 7 / mL to obtain a protoplast suspension.

[0033] Add the fragment UngoD-hph-DngoD to 150 μL of the above protoplast suspension, then add 50 μL of PSTC, mix gently, and place on ice for 30 minutes. Add 1 mL of PSTC, mix well, and place at room temperature for 20 minutes. Finally, mix with 10 mL of top agar and pour on 3 0.8 M PDAS-H plates, and culture at 30°C in the dark for 5 days to obtain transformants.

[0034] Transformants with hygromycin resistance were picked from the 0.8M PDAS-H transformation screening plate and transferred to the PDA-H plate, and cultured at 25°C for 5 days. The genome of each transformant was extracted and PCR was performed using external primers U-ngoD-F / D-ngoD-R. The positive transformants were those that could amplify a band of 4520 bp, while the control strain MKAJ could only amplify a band of about 2.9 kb ( Figure 1 ).

[0035] The transformants were cultured on PDA-H plates, with MKAJ as the control strain, at 25°C for 5 days. A small amount of mycelium was picked and extracted using a nucleic acid extraction instrument ( -24) The mycelium was crushed and inoculated into 50mL ME seed culture medium (250mL Erlenmeyer flask), and cultured on a shaking table at 25°C, 220rpm for 45-48h. 5mL of the above-cultured seed solution was added to 50mL ME fermentation medium (250mL Erlenmeyer flask), and cultured on a shaking table at 25°C, 220rpm for 8 days, with 3 parallels for each strain. After the fermentation was completed, 90mL of ethyl acetate was added to 50mL of the fermentation broth, and the mixture was extracted at 25°C and 220rpm for 1h, and allowed to stand for 10min. The upper organic phase was taken for vacuum distillation, and after evaporation, 2mL of chromatographic methanol was added to dissolve the crude extract, and then the treated sample was filtered with a 0.22μm organic filter, and the treated sample was analyzed by HPLC.

[0036] The HPLC analysis method is as follows: the liquid chromatography column is Agilent C-18 reverse phase column 883975-902 (4.6×150 mm, 5 μm); mobile phase A: 0.05% (volume ratio) trifluoroacetic acid acetonitrile solution, mobile phase B: 0.05% (volume ratio) trifluoroacetic acid aqueous solution; the flow rate is 1 mL / min, the ultraviolet detection wavelength is 210 nm, 30° C., and the total elution time is 33 min. Gradient elution conditions: 0-25 min, the volume of mobile phase A accounts for a linear increase from 5% to 100% of the mobile phase; 25-30 min, the volume of mobile phase A accounts for 100% of the mobile phase, and 30.1-33 min, the volume of mobile phase A accounts for 5% of the mobile phase.

[0037] The results are as follows Figure 2 As shown, after knocking out the gene ngoD, the anthraquinone compound final product 1 was not produced at all, but an anthraquinone compound intermediate 2 was produced. It is speculated that the gene ngoD is responsible for methylating intermediate 2 to generate the final product 1.

[0038] 2. In vitro activity verification of methyltransferase NgoD

[0039] The function of gene ngoD was verified by in vitro enzyme activity. First, gene ngoD was synthesized and connected to expression vector pET28a, and then induced expression and purified.

[0040] The E. coli puncture bacteria containing the ngoD expression vector were inoculated into 5 mL of LB medium containing a final concentration of 50 μg / mL kanamycin, cultured overnight at 37°C for 18 hours, and the plasmid was extracted using the OMEGA plasmid extraction kit. The quality of the plasmid extraction was detected by 1% agarose gel electrophoresis. 1 μL of the plasmid was transformed into BL21 (DE3), and the growing transformants were picked on the kanamycin resistance plate and cultured in 10 mL of liquid LB medium containing a final concentration of 50 μg / mL kanamycin at 37°C for 18 hours, and then transferred to 1L of LB medium with a final concentration of 50 μg / mL kanamycin at 37°C to OD 0.6-0.8, and IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.2 mM at 16°C to induce protein expression for 20 hours, and the bacteria were collected by centrifugation at 5000×g.

[0041] Resuspend the cells with 20 mL of lysis buffer, place them under an ultrasonic disruptor for 30 minutes (5 seconds on / 5 seconds pause), and then collect the supernatant of the disrupted liquid by centrifugation at 10,000 rpm for 60 minutes. Add 1 mL of Ni-NTA Agarose res in to the supernatant, rotate and shake at 4°C for 2 hours, then slowly fill the Ni-NTA Agarose res in into an empty column and wait for the liquid to flow out. Next, use 100 mL of rinse buffer to slowly wash away non-specifically bound impurities. Thereafter, elute the bound target protein with 10 mL of elution buffer. Finally, ultrafiltration, concentration and desalting were performed using an Amicon Ultra-15 ultrafiltration tube with a molecular weight cutoff of 30 kDa. All of the above purification steps were performed at 4°C.

[0042] like Figure 3 As shown, SDS-PAGE analysis showed that the target protein ngoD had been successfully expressed and purified and could be used for the next in vitro activity analysis study.

[0043] The obtained target protein NgoD was used to study the catalytic activity of the enzyme when its own substrate compound 2 was used as a substrate. The reaction system is shown in Table 1.

[0044] Table 1 In vitro reaction system with compound 2 as substrate

[0045]

[0046]

[0047] After reacting at 30°C and 160 rpm for 2 hours, 400 μL of ethyl acetate was added to stop the reaction; after vigorous shaking for 30 minutes, the supernatant ethyl acetate phase was taken and dried with nitrogen; then 200 μL of methanol was added and vigorously shaken for 10 minutes to fully dissolve the product; then centrifuged at 13000 rpm for 10 minutes to precipitate impurities. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC.

[0048] The results are as follows Figure 4 As shown, NgoD can catalyze the reaction of compound 2 to produce a compound with an HPLC retention time consistent with that of the standard compound 1, indicating that NgoD has methyltransferase activity.

[0049] Since the structure of compound 2 is similar to that of rhamnosin, in order to verify whether NgoD can methylate rhamnosin to produce rhamnosin methyl ether in vitro, the obtained protein NgoD was used to study the catalytic activity of the enzyme when rhamnosin was used as a substrate. The reaction system is shown in Table 2.

[0050] Table 1 In vitro reaction system with rhein as substrate

[0051] system concentration Substrate: Rhein 100μM Donor: SAM 1mM Enzyme: NgoD protein 20μM Buffer: Tris-HCl (pH 7.5, make up to 100 μL) 20mM

[0052] After reacting at 30°C and 160 rpm for 2 hours, 400 μL of ethyl acetate was added to stop the reaction; after vigorous shaking for 30 minutes, the supernatant ethyl acetate phase was taken and dried with nitrogen; then 200 μL of methanol was added and vigorously shaken for 10 minutes to fully dissolve the product; then centrifuged at 13000 rpm for 10 minutes to precipitate impurities. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC.

[0053] The results are as follows Figure 5 As shown, NgoD can catalyze the reaction of rhamnosin to produce a compound whose HPLC retention time is consistent with that of rhamnosin-3-methyl ether standard.

[0054] 3. Heterologous expression of methyltransferase NgoD in Aspergillus terreus

[0055] In order to verify whether NgoD can methylate rhamnosin to produce rhamnosin-3-methyl ether in vivo, heterologous expression was used to verify it.

[0056] Aspergillus terreus HZ04 with high homologous recombination efficiency was selected as the chassis strain, and the gene ngoD was inserted into its gedA gene (the protein sequence encoded by it is shown in SEQ ID NO: 3, which is responsible for the methylation modification of the C1 hydroxyl group of rhamnosus). Figure 6The homologous recombination strategy shown in the figure uses ptrA as a resistance screening tag. Based on this strategy, three expression elements were constructed: UgedA-ngoD-Tpgk-ptrA-DgedA, UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedA and UgedA-PgpdAt-ngoD-Tpgk-ptrA-DgedA. (The nucleic acid sequence of the PgedA promoter is shown in SEQ ID NO:4, the nucleic acid sequence of the PgpdA promoter is shown in SEQ ID NO:5, and the nucleic acid sequence of the PgpdAt promoter is shown in SEQ ID NO:6).

[0057] The constructed expression element was tested by PCR, and the results were as follows Figure 7 As shown, the amplified expression element UgedA-ngoD-Tpgk-ptrA-DgedA is close to the theoretical size of 5037bp, and the size is correct; the amplified expression element UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedA is close to the theoretical size of 6172bp, and the size is correct; the amplified expression element UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedAt is close to the theoretical size of 5783bp, and the size is correct.

[0058] The expression element was purified. Since the amplified targeting elements all had mixed bands, they were purified by gel recovery to obtain the purified expression element.

[0059] Using Aspergillus terreus HZ04 as the starting strain, first take an appropriate amount of spore suspension from the 1.2M PDAS plate, inoculate it into 50mL of YPM seed culture medium, and culture it in a 250mL Erlenmeyer flask at 220rpm and 30℃ in a shaking table. After 2 days, collect the mycelium with a 300-mesh filter cloth. Wash the mycelium 5 times with 0.6M MgSO4. Weigh 1g of mycelium, add 10mL of AT enzymatic hydrolyzate (filtered and sterilized through a 0.22μm sterile filter), and treat it at 30℃ and 140rpm for 2h. Filter the above protoplast reaction liquid through a 500-mesh filter cloth and a sterile magic filter cloth, and collect the protoplasts by centrifugation at 5000rpm and 4℃. Wash once with precooled STC, resuspend the protoplasts in precooled STC, and adjust the protoplast concentration to 10 with STC. 7 / mL to obtain a protoplast suspension.

[0060] Add 150 μL of the above protoplast suspension to the three purified expression elements, then add 50 μL of PSTC, mix gently, and place on ice for 30 minutes. Add 1 mL of PSTC, mix well, and place at room temperature for 20 minutes. Finally, mix with 10 mL of top agar, pour on 3 CD-P plates, and culture at 28°C in the dark for 5 days to obtain transformants.

[0061] Transformants with pyrithiamine resistance were selected from the CD-P transformation screening plate and transferred to new CD-P plates and cultured at 28°C for 5 days. The genome of each transformant was extracted and verified by PCR using external primers U-gedA-F / D-gedA-R. The results were as follows: Figure 8 As shown, multiple positive transformants were obtained for all three expression elements.

[0062] Positive transformants were selected and cultured in 1.2M PDAS plates (28°C, 5 days) for conidia. After the culture was completed, sterile water was added to wash out the spores to obtain a spore suspension, which was then inoculated with 10 7 spores to 50mL LPM fermentation medium (250mL Erlenmeyer flask), 28℃, 220rpm, shaker culture for 8 days, set up 3 parallels for each strain. After the fermentation, take 1mL of fermentation broth, centrifuge at 12000rpm for 10min, retain the bacteria, add the bacteria to 10mL methanol: dichloromethane (1:1) mixed solution and rotate for 2h, stand for 10min, separate the methanol: dichloromethane (1:1) mixed solution from the bacteria, and collect the mixed solution. Take 500μL of the mixed solution, blow dry with nitrogen, add 500μL of methanol, shake and dissolve for 10min, filter with 0.22μm organic filter membrane, and perform HPLC analysis. The HPLC analysis method is: use Agile 1260HPLC to analyze the fermentation extract on Agile Eclepse Plus C18 column (5μm, 4.6mm×250mm). The analysis method is as follows: mobile phase A (100% H2O + 0.05% TFA), mobile phase B (100% ACN + 0.05% TFA), gradient elution (0-1min 50% A, 1-20min 50%-0% A, 20-24min 0% A, 24-25min 0%-50% A, 25-30min 50% A), flow rate 1mL / min, detection wavelength 440nm. The results show:

[0063] The gedA gene is responsible for catalyzing the methylation modification of the C1 hydroxyl group of rhubarb. After the gedA gene was knocked out, the methylation modification of the C1 hydroxyl group of rhubarb could not be carried out, resulting in the accumulation of rhubarb. Since the starting strain HZ04 did not have the gedA gene knocked out, there was no accumulation of rhubarb, which was used as a control; in addition, the standards of rhubarb and rhubarb-3-methyl ether were used as controls. From the analysis results, it can be seen that the HZ04-PgedA-ngoD transformant produced rhubarb and a trace amount of rhubarb methyl ether, indicating that the gedA gene was successfully knocked out, and the gedA promoter can drive the gene ngoD to function, but the transformation efficiency is low; rhubarb accumulated in the HZ04-PgpdA-ngoD transformant, and rhubarb methyl ether was almost undetectable. The HZ04-PgpdAt-ngoD transformant produced rhubarb and rhubarb methyl ether, and the accumulation of rhubarb methyl ether was higher than the previous two.

[0064] Therefore, it can be concluded that the methyltransferase NgoD can catalyze rhamnosin to produce rhamnosin-3-methyl ether in Aspergillus terreus.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A methyltransferase capable of catalyzing the methylation of anthraquinone compounds, characterized in that: The amino acid sequence is shown in SEQ ID NO:

2.

2. A nucleic acid encoding the methyltransferase according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The sequence is shown in SEQ ID NO:

1.

4. Use of the methyltransferase according to claim 1 in methylating anthraquinone compounds.

5. A method for preparing methylated anthraquinone compounds, characterized in that: The method comprises the steps of mixing the anthraquinone compound to be methylated with the methyltransferase and a methyl donor as claimed in claim 1 and performing a catalytic reaction.

6. The method according to claim 5, characterized in that The catalytic reaction is carried out in vitro.

7. The method according to claim 5, characterized in that The catalytic reaction is carried out in vivo by expressing the methyltransferase in cells containing the anthraquinone compounds to be methylated.

8. The method according to claim 7, characterized in that The methylated anthraquinone compound is rhein-3-methyl ether, and the cell is a cell capable of accumulating rhein.

9. The method according to claim 8, characterized in that The cell is Aspergillus terreus capable of accumulating rhein.

10. A cell capable of producing methylated anthraquinone compounds, characterized in that: It is prepared by the method according to any one of claims 7 to 9.

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